Method and compositions for enhancing aminolevulinic acid dehydratase assay

ABSTRACT

The present invention provides a method of improving the sensitivity and accuracy of a lead assay. The method enhances the recovery of lead during isolation of the lead from interfering compounds by maintaining the lead in a sample solution and making the recovered lead available for detection by the assay. An enhancing reagent complexes with the lead isolated in the sample solution. The enhancer includes a chelator having a lead equilibrium binding constant in the range of about 4 log K to about 13 log K. A kit for performing such a lead assay is also provided.

This application is a continuation of application Ser. No. 08/171,121,filed Dec. 21, 1993 now abandoned.

RELATED APPLICATION

Related U.S. application Ser. No. 08/171,035 filed on even date herewithnow abandoned discloses certain reagents which are suitable for use inthe present application. The entire teaching and disclosure of thatco-pending application is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to assays for detecting metal ions such aslead and, more particularly, utilizing selected chelators to enhance thesensitivity and accuracy of a whole blood lead assay usingaminolevulinic acid dehydratase.

BACKGROUND OF THE INVENTION

The rapid determination of trace metals in biological and environmentalsystems is increasingly important in identifying potential hazards andpreserving the public health. The toxicity of certain metals such aslead is well-known. The absorption of even trace amounts of lead cancause severe damage to human organs. The numerous and widespread sourcesof lead in the environment, including the food supply, compounds theproblems of screening affected groups.

It is generally recognized that lead poisoning occurs in children atblood levels as low as 10-15 ug/dl. Lead contamination of environmentalsources such as water, dust and soil require identification at evenlower levels. To measure these amounts, the analytical techniques mustbe sensitive, contaminant-specific, and reliable.

An early technique for identifying trace metals in biologic systems isdescribed by J. Pierce et al., "Lead, Chromium, and Molybdenum by AtomicAbsorption, Arch. Environ. Health, 13:209 (1966). This measurementtechnique requires extraction of the lead from blood samples byprecipitating the blood proteins with trichloroacetic acid andcomplexing the lead with a chelator under acidic conditions. The metalcomplex was then analyzed by atomic absorption. The skill and expensiveinstrumentation required by this technique limits its applicability fordetecting lead contamination.

The use of d-aminolevulinic acid dehydratase (ALAD) activity in redblood cells to determine exposure to environmental lead is described byA. Berlin, et al., "European Standardized Method for the Determinationof d-Aminolevulinic Acid Dehydratase Activity in Blood," Z. Klin. Chem.Klin. Biochem., 12 Jg. 1974, S. 389-390. The assay entails incubation ofthe enzyme with excess d-aminolevulinic acid (ALA). The porphobilinogen(PBG) which is formed within a fixed time is mixed with modifiedEhrlich's reagent, and the color developed is measured photometricallyagainst a blank. The quantity of PBG produced is a measurement of theALAD activity and corresponds to the low levels of lead exposure.

A method of colormetric determination of ALAD is presented by S. Sassa,"Delta-Aminolevulinic Acid Dehydratase Assay," Enzyme, 28:133-145(1982). The effect of reducing agents like dithiothreitol (DTT) on theenzyme activity and its use for the detection of subclinical leadpoisoning by assaying the enzyme activity in erythrocytes is disclosed.

Two articles, each entitled "Purification and Properties ofd-Aminolevulinate Dehydrase from Human Erythrocytes," first published byP. Anderson, et al., J. Biol. Chem., 254:6924-6930 (1979) andsubsequently by P. Gibbs, et al., Biochem J., 230:25-34 (1985), discloseassays demonstrating lead as a noncompetitive inhibitor of ALADactivity. The incubation mixtures contained DTT, ALAD and ALA in abuffer solution. The incubations were terminated by the addition of TCAcontaining HgCl₂. The solution was centrifuged and the supernatant wasadded to modified Ehrlich's reagent in acetic acid and HClO₄. Thecolored complex formed with PBG was measured spectrophotometrically.

A similar assay measuring the activity of ALAD after exposure to leadcontaining samples is disclosed in a published PCT application WO93/01310 to Silbergeld. The application suggests utilizing otherwell-know methods like conjugating or attaching a label to either thesubstrate or product and quantifying the amount of labeled materialpresent after a defined reaction period. Another approach suggested usesa known antibody that binds specifically to unoccupied lead bindingsites of ALAD.

A significant problem in using an ALAD assay is the poor lead recoveryin the preparation of a sample. For an ALAD assay a sample of wholeblood is typically pretreated with acid to release lead from the redblood cells and to precipitate sample proteins. The pretreated sample isthen neutralized before the enzyme is added for incubation. Usually, therecovery of lead is less than the lead-spiked water control sample.

Effective screening of potentially affected people or sources demands ananalytical technique which is conveniently used, inexpensive andconvenient both as to the sample size and administration of the test.For early detection, contamination prevention, or corrective treatment,the time interval between sampling and results is preferably minimal.Although the lead assay using the enzyme ALAD has proven useful, thereis a need to enhance the sensitivity and accuracy. The present inventionprovides enhancing reagents and methods which improve the sensitivity ofthe assay by recovering more of the lead in the sample duringpretreatment and neutralization makes it available to inhibit theactivity of the ALAD enzyme.

SUMMARY OF THE INVENTION

The present invention provides a method of improving the sensitivity andaccuracy of a lead assay. The method enhances the recovery of leadduring isolation of the lead from interfering compounds by maintainingthe lead in a sample solution and making the recovered lead availablefor detection by the assay.

The method includes isolating the lead in a sample solution fromcompounds which interfere with the lead assay. The recovery of the leadis enhanced in the sample solution and the lead is made available forassay. An ALAD enzyme is incubated in the sample solution in thepresence of a substrate. The enzyme incubation step is stopped after apredetermined time interval. The extent of the enzyme activity issubsequently photometrically determined.

The present invention also provides an enhancing reagent for enhancingthe recovery of lead in a test sample and making the lead available toimprove the sensitivity and accuracy of a lead assay. The enhancercomprises a chelator having a lead equilibrium binding constant in therange of about 4 log K to about 13 log K. A kit for performing such alead assay is also provided.

Accordingly, it is an advantage of the present invention to provide amore sensitive and accurate lead assay using the ALAD enzyme.

Another advantage of the present invention is to provide a method ofrecovering more lead after isolation of the lead in a sample and make itavailable for detection in an assay.

A further advantage of the present invention is to provide an enhancerreagent which has a minimal adverse effect on the other steps of theassay.

Other and further advantages, embodiments, variations and the like willbe apparent to those skilled in the art from the present specificationtaken with the accompanying drawings and appended claims.

DETAILED DESCRIPTION

The present invention relates to the enhancement of the sensitivity ofan assay for determining lead by exposing an ALAD enzyme to a samplesuch as whole blood and measuring the inhibition of activity.Preparation for the assay includes pretreating the sample to expose andrecover the lead from within the red blood cells and to precipitateinterfering compounds such as proteins, endogenous ALAD, PBG, ALA andthe like. Acid is commonly used to pretreat the sample. The interferingcompounds are then pelleted by centrifugation leaving a supernatantwhich contains the lead isolated from the interfering compounds.

The acidified supernatant containing the recovered lead is thenseparated for additional processing. The supernatant must be neutralizedprior to incubation of the ALAD enzyme. A neutralizing reagent is addedto bring the supernatant sample to a neutral pH. During thisneutralization step, it is important to maintain the lead present in thesupernatant sample in an enzyme accessible form which enhances thesensitivity and accuracy of the assay by increasing the amount of leadwhich is recovered from the sample and available for enzyme detection.

The present invention improves the recovery of lead from the sample byadding an enhancing reagent which keeps the lead in the supernatantsample during the neutralization step. The enhancer forms a complex orcompound with the lead released in the supernatant sample. Theenhancer-lead complex maintains the lead in the supernatant sample andavoids its loss due to precipitation or the like. The enhancer must thenmake the lead available for inhibiting the activity of the ALAD enzyme.The enhancer reagent also must avoid or minimize any interference,directly or indirectly, with the activity of the ALAD enzyme. Indirectinterference, for example, results from affecting the concentration ofzinc in the assay. Having the enhancer reagent form a complex with zincdecreases the activity of the ALAD enzyme.

As used herein, the term "enhancer" means a reagent provided by thepresent invention that increases the accuracy and sensitivity of a leadassay in comparison to that achieved by the assay in the absence of thereagent. The enhancer forms a complex or compound with the releasedlead, maintains availability of the lead in the sample and then allowsit to inhibit the ALAD enzyme activity. The term "neutralizing reagent"is the solution which brings the acidified supernatant sample to aneutral pH.

The present invention has found that certain chelating reagents complexwith the sample lead and still make it available to inhibit the activityof the ALAD enzyme. The currently preferred enhancers for use with thepresent invention are N-benzyliminodiacetic acid (BenzylIDA),L-histidine monohydrochloride monohydrate (Histidine),N-(2-hydroxyethyl)-iminodiacetic acid (HEIDA), iminodiacetic acid (IDA),DL-penicillamine (PEN), methyliminodiacetic acid (MIDA),nitrilotriacetic acid (NTA), sodium citrate, andd-hydroxyquinoline-5-sulfonic acid hydrate (HQSA).

The present invention has also found that enhancers which exhibit acharacteristic to form a complex with Pb⁺² as described by the Pb⁺²equilibrium binding constant (K) in the range of about 4 log K to about13 log K are suitable enhancers. Preferably, the enhancer exhibits aPb⁺² equilibrium binding constant in the range of about 6 log K to about9 log K.

All the of preferred enhancers identified above have a Pb⁺² equilibriumbinding constant within the suitable range. Other enhancers whichsimilarly exhibit a suitable Pb⁺² equilibrium binding constant include8-hydroxy-5-(2'-hydroxyphenylazo) quinoline, 8-hydroxy-5-(phenylazo)quinoline, N-(2-carboxyphenyl)iminodiacetic acid,N-(acetonyl)iminodiacetic acid, N-(dithiocarboxy)aminoacetic acid,N,N-bis(2'-hydroxyethyl)glycine, and glycine.

Other enhancers contemplated by the present invention includedihydroxyphenyl acetic acid, N-(2'carboxyethyl) iminodiacetic acid,dihydroxybenzoic acid, 3,4,dihydroxybenzene sulfonic acid, melonic acid,1-hydroxy-1-(3'-pyridyl) methane sulfonic acid, and4-aminopyridine-2,6-dicarboxylic acid.

The present invention contemplates a method of enhancing the sensitivityand accuracy of a lead assay by isolating the lead in a sample solutionfrom compounds which interfere with the lead assay. The pretreatment ofthe sample can be accomplished with conventional techniques such as byadding TCA, nitric acid, 5-sulfosalicylic acid, or perchloric acid tothe whole blood sample.

The acidified sample must then be neutralized before the assay cancontinue with incubation of the ALAD enzyme in its presence. To preventprecipitation of the lead or the formation of a lead complex which isnot detectable by the assay, the enhancer is present in the sampleduring adjustment of the sample's pH to neutral. The enhancer recoversthe lead in the sample solution and makes the lead available forcontinuing the assay.

The assay continues by incubating an ALAD enzyme in the sample solutionin the presence of a substrate such as ALA. The enzyme incubation stepis stopped after a predetermined time interval. The product of theenzyme activity is PBG. By reacting the PBG with Erhlich's reagent toform a chromophore, one can photometrically determine the extent of theenzyme activity. Other coloring reagents containingdimethylaminobenzaldehyde, dimethylaminocinnamaldehyde or theirderivatives are suitable for use with the present invention.

The following Examples are set forth for the purposes of illustrationand should not be construed as limiting.

EXAMPLES Materials

All the reagents used in the present invention are availablecommercially. The aminolevulinic acid (ALA) and d-aminolevulinic aciddehydratase (ALAD) were both purchased from Sigma Company of St Louis,Mo., as cat. nos. A-3785 and A-0644, respectively. The HgCl₂,dimethylaminobenzaldehyde (DMAB) and dithiothreitol are also availablefrom Sigma as cat. nos. M-6529, D-8904 and D-0632. The buffer (bis2-hydroxyethyl!imino-tris hydroxymethyl!methane (BisTris), concentratedHNO₃ acid, NaOH pellets, ZnCl₂ (99+% pure), and trichloroacetic acid(TCA) are available from Aldrich Chemical Company of Milwaukee, Wis., ascat. nos. 15,666-3, 22,571-1, 30,657-6, 22,999-7, and 25,139-9. The 10mg/dl lead standard was also purchased from Aldrich as cat. no.31,903-3. The glacial acetic acid and 60% perchloric acid were purchasedfrom Fisher as cat. nos. A38-212 and A228-1

The enhancers tested include: N-benzyliminodiacetic acid (BenzylIDA)purchased from Aldrich as cat. no. B2,475-8 (98% pure); ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) Aldrich cat. no. 23,453-2(97% pure); ethylenediaminetetraacetic acid (EDTA) purchased from Sigmacat. no. ED2P (98% pure); L-histidine monohydrochloride monohydrate(Histidine) purchased as Aldrich cat. no. H1,520-9 (98% pure);N-(2-hydroxyethyl)-iminodiacetic acid (HEIDA) purchased as Aldrich catno. 15,814-3 (98% pure); Iminodiacetic Acid (IDA) purchased from Aldrichcat. no. I-120-0 (97% pure); DL-penicillamine (PEN) purchased fromAldrich cat. no. P60-8 (99+% pure); methyliminodiacetic acid (MIDA)purchased from Aldrich cat. no. M5,100-8 (99% pure); nitrilotriaceticacid (NTA) purchased from Aldrich cat no. 10,629-1 (99+% pure); sodiumcitrate purchased from Mallinckrodt cat. no. 0754 (99.7% pure);d-hydroxyquinoline-5-sulfonic acid hydrate (HQSA) purchased from Aldrichcat. no. H5,875-7 (98% pure).

The spectrophotometer used for absorbance measurements was a LKBUltraspec II Model 4050.

Method

All flasks used in the preparation of solutions described herein werewashed with 1 N HNO₃. The solutions prepared were stored at roomtemperature unless otherwise noted. The pH of 50 ml of HPLC gradedistilled water was adjusted to a pH 1.50 by adding an appropriateamount of concentrated HNO₃. A 20 mM ZnCl₂ solution was prepared byadding pH 1.50 distilled water to 0.0340 g. ZnCl₂ for a final solutionweight of 12.500 g. The solution was then thoroughly mixed.

A solution of 200 ml 1.5 M BisTris was prepared by adding 62.70 g. ofBisTris to HPLC grade distilled water to a final volume of about 180 ml.After stirring, the pH was adjusted to 7.30 with concentrated HNO₃. Theresulting volume was adjusted to the mark with distilled water.Similarly a 200 ml solution of 2.0 M BisTris was prepared by using 83.60g. of BisTris. The pH was adjusted to pH 7.60 before adjusting thevolume. Both BisTris solutions were stirred for 10 min. at roomtemperature and filtered to remove any visible particles.

A diluted enzyme reagent was prepared by adding 5 ml of ALAD containing3.1 U/mg to 35 ml of 250 mM BisTris. The 250 mM BisTris diluent solutionwas prepared by adding 5.23 g. BisTris to 100 ml of HPLC grade distilledwater and stirring. DTT was added to 10 mM in the diluted enzymereagent. Other sulfhydryl compounds such glutathione, mercaptoethanoland cysteine can be used as a reducing agent instead of DTT. The pH ofthe diluent solution was adjusted to pH 7.0 by adding 50% NaOH. Thediluted enzyme reagent was stored at 2°-8° C. under nitrogen gas.

A 25 mM ALA and 10 uM ZnCl₂ substrate solution was prepared by adding0.210 g. ALA, 25 ul 20 mM ZnCl₂ and 50 ml HPLC distilled water to aflask. After stirring, the substrate solution was stored at 2.8° C. inthe dark.

A stop reagent containing 10% TCA was prepared by adding 20.000 g ofTCA, 0.1 M HgCl₂ and HPLC grade distilled water to 200 ml. The solutionwas stirred and filtered at 0.80 um.

A pretreatment reagent containing 17.5 % TCA was prepared by adding 17.5g TCA to 100 ml HPLC distilled water and stirring.

A modified Ehrlich's Reagent was prepared by adding 12.5 g DMAB, 250 mlglacial acetic acid and 122.5 ml of 60% perchloric acid and mixing. Thefinal volume was adjusted to 500 ml by adding more glacial acetic acid.The modified Ehrlich's Reagent was stored in the dark at 2°-8° C.

A whole blood mixture was prepared by collecting 180 ml of whole bloodfrom type B+ and O+ donors and adding 20 mg of sodium heparin. A testsample containing 40 ug/dl Pb⁺² was prepared by adding 0.320 g of 10mg/dl Pb⁺² to 80.0 ml of the whole blood mixture. The remainder of thewhole blood mixture was used as a control containing 0 ug/dl of Pb⁺².After vigorous mixing, both test samples were stored for 4 hours withmixing every 30 min., and subsequently, overnight at 2°-8° C.

For each enhancer, a neutralizing solution containing 0.5 M enhancer and1.5 M BisTris was prepared by adding 7.5 ml of the 2 M Bis-Tris solutionto following amounts of enhancers Na Citrate 1.470 gm; IDA 0.975 g; NTA1.175 g; EGTA 1.900 g; Histidine 1.050 g; HEIDA 0.885 g; MIDA 0.735 g;BenzylIDA 1.115 g; HQSA 1.125 g; EDTA 1.840 g; EDTA 1.840 g; and PEN0.745 g. After stirring, HPLC grade distilled water was added to eachneutralizing solution to obtain a final volume of about 9.5 ml.Subsequently, the enhancer solutions were vigorously stirred overnight.The neutralizing solutions containing Histidine, HQSA and PEN enhancerreagents were heated to about 70° C. to complete dissolution and thencooled to room temperature. The pH of each neutralizing solution wasthen adjusted to pH 7.25 with either concentrated HNO₃ or 50% NaOH.

Whole blood samples containing 0 ug/dl and 40 ug/dl Pb⁺² were dispensedin 24.5 ml amounts and were pretreated with 10.5 ml of the TCApretreatment solution. Each sample was centrifuged for five minutes andthe supernatants were saved. From each supernatant solution 180 ul wasmixed by vortex with 180 ul of neutralizing buffer. From thisneutralized supernatant solution 100 ul was added to 100 ul of thedilute enzyme reagent and mixed by vortex and incubated for 15 min in a37° C. water bath.

Subsequently 100 ul of the substrate solution was added, mixed by vortexand incubated for 30 min. in the water bath. The stop reagent was addedin an amount of 250 ul and mixed by vortex. The mixture was centrifugedfor two minutes to remove insoluble DTT.

The supernatant mixture was removed and added to 500 ul of the modifiedEhrlich's reagent. After mixing by vortex and incubating for 10 min. atroom temperature, the absorbance was recorded at 555 nm.

Results

The absorbance data for each enhancer was collected at the followingconcentrations, measured in mM after being mixed with the pretreatedwhole blood supernatant:0.25, 1.00, 2.50, 10.00, 25.00, 100.00 and250.00. Table 1 shows the measured absorbance span for each enhancer inthe samples containing 0 ug/dl and 40 ug/dl Pb⁺². The equilibriumconstant of Pb⁺² for each enhancer as reported in either Bjerrum, supra,or Sillen, supra, is included in Table 1.

                  TABLE 1                                                         ______________________________________                                                              Absorbance                                                                              Pb.sup.+2  Equilibrium                        Enhancer   Conc. mM   Span      Constant log (Km)                             ______________________________________                                        Control    --         0.006     --                                            Na Citrate 250.00     0.106     4.3                                           Histidine  250.00     0.915     6.4                                           IDA        250.00     0.801     7.5                                           MIDA       100.00     0.735     8.0                                           HQSA       2.50       0.891     8.5                                           HEIDA      250.00     0.223     9.5                                           NTA        10.00      0.110     11.6                                          Penicillamine                                                                            100.00     0.311     13.0                                          EGTA       10.00      0.016     14.7                                          ______________________________________                                    

As a matter of convenience, the reagents can be provided as kits, wherethe reagents are in predetermined ratios, so as to substantiallyoptimize the sensitivity of assay in the range of interest. Wet or dryreagents may be used. After reconstitution of dry reagents, inpredetermined volumes, the concentration of the reagents will be atappropriate levels.

The reagents may be mixed with various ancillary materials such asneutralizing solutions, buffers, substrate solutions, and the like. Themethod of the present invention is performed by combining certain of thethree reagents in a mixture essential for the pretreatment of the leadsample. One is the acidified lead sample, the second is the neutralizingreagent and the third is the enhancer. The enhancer may be kept separateor added to either the neutralizing solution or the acidified leadsample.

As demonstrated above, the present invention provides chelating reagentsto enhance the sensitivity and accuracy of a lead assay using the ALADenzyme. More of the lead in the sample is recovered and detected ascompared to using the assay in the absence of the enhancer.

Although the invention has been described using a photometric analysisof the PBG to determine lead contamination, the present invention is notso limited. The method and compositions of the present invention can beemployed in various heterogeneous and homogeneous immunoassay systemformats known in the art. Such immunoassay system formats include, butare not intended to be limited to, competitive, sandwich andimmunometric techniques. Generally, such immunoassay systems depend uponthe ability of a binding member, such as, for example, an immunoglobulin(i.e., a whole antibody or fragment thereof) to bind to a specificanalyte from a test sample, wherein a labeled reagent comprising abinding member labeled with a signal generating compound such as afluorescent or chemiluminescent molecule is employed to determine theextent of binding. Typically, the extent of binding in such immunoassaysystem formats is determined by the amount of the signal generatingcompound present in the labeled reagent which either has or has notparticipated in a binding reaction with the analyte, wherein the signalwhich is generated by the signal generating compound as described hereinis detected and correlated to the amount of analyte present in the testsample. Accordingly, the amount of analyte is correlated to the level oflead contamination of the test sample.

Homogeneous immunoassays typically are performed in a competitiveimmunoassay format involving a competition between an analyte from atest sample and a labeled reagent for a limited number of receptorbinding sites on an antibody to the analyte. The labeled reagentcomprises the analyte or analyte-analog labeled with a signal generatingcompound wherein the concentration of analyte in the test sampledetermines the amount of the labeled reagent that will specifically bindto the antibody. The amount of the labeled reagent-antibody conjugateproduced by such binding may be quantitatively measured and is inverselyproportional to the amount of analyte present in the test sample.

Heterogeneous immunoassay formats involve a labeled reagent or tracercomprising an analyte, analyte-analog, or an antibody thereto, labeledwith a signal generating compound, to form a free species and a boundspecies. In order to correlate the amount of tracer in one of suchspecies to the amount of analyte present in the test sample, the freespecies must first be separated from the bound species, which can beaccomplished according to methods known in the art employing solid phasematerials for the direct immobilization of one of the bindingparticipants in the binding reaction, such as the antibody,analyte-analog, or analyte, wherein one of the binding participants isimmobilized on a solid phase material, such as a test tube, beads,particles, microparticles or the matrix of fibrous material, and thelike, according to methods known in the art. The solid phase materialscan be any solid material to which a binding participant can beimmobilized and include, but are not intended to be limited to, beads,magnetic particles, paramagnetic particles, microparticles or macroparticles, test tubes, and microtiter plates. Such solid phase materialscan be made from synthetic materials, naturally occurring materials, ornaturally occurring materials which have been synthetically modified,and include, but are not intended to be limited to, cellulose materials,such as paper, cellulose and cellulose derivatives such as celluloseacetate and nitrocellulose; fiberglass; naturally occurring cloth suchas cotton; synthetic cloth such as nylon; porous gels, such as silica,agarose, dextran, and gelatin; porous fibrous matrixes; starch basedmaterials, such as cross-linked dextran chains; ceramic materials;olefin or thermoplastic materials including polyvinyl chloride,polyethylene, polyvinyl acetate, polyamide, polycarbonate, polystyrene,copolymers of vinyl acetate and vinyl chloride, combinations ofpolyvinyl chloride-silica; and the like.

Heterogeneous immunoassays can be performed in a competitive immunoassayformat wherein, for example, the antibody can be immobilized to a solidphase material whereby upon separation, the signal generated by thesignal generating compound of the bound or free species can be detectedand correlated to the amount of analyte present in the test sample.Another form of a heterogeneous immunoassay employing a solid phasematerial is referred to as a sandwich immunoassay, which involvescontacting a test sample containing, for example, an antigen with aprotein such as an antibody or another substance capable of binding theantigen, and which is immobilized on a solid phase material. The solidphase material typically is treated with a second antigen or antibodywhich has been labeled with a signal generating compound. The secondantigen or antibody then becomes bound to the corresponding antigen orantibody on the solid phase material and the signal generated by thesignal generating compound in the bound or the free species can bedetected and correlated to the amount of analyte present in the testsample.

The present invention is also not limited to the analysis of leadcontamination in a blood sample. The test sample can be derived from anybiological source, such as a physiological fluid, including, blood,saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk,ascites fluid, raucous, synovial fluid, peritoneal fluid, amniotic fluidor the like. The test sample can be pretreated prior to use, such aspreparing plasma from blood, diluting viscous fluids, or the like;methods of treatment can involve filtration, distillation,concentration, inactivation of interfering components, and the additionof reagents. Besides physiological fluids, other liquid samples can beused such as water, food products and the like for the performance ofenvironmental or food production assays. In addition, a solid materialsuspected of containing the analyte can be used as the test sample. Insome instances it may be beneficial to modify a solid test sample toform a liquid medium or to release the lead. Generally, any samplesuspected of containing lead can be analyzed as long as the lead isliberated from the physical or chemical mixture in which it is presentedto produce elemental lead.

While particular embodiments and applications of the present inventionhave been illustrated and described, it is to be understood that theinvention is not limited to the precise construction and compositionsdisclosed herein and that various modifications, changes, and variationswhich will be apparent to those skilled in the art may be made in thearrangement, operation, and details of construction of the inventiondisclosed herein without departing from the spirit and scope of theinvention as defined in the appended claims.

What is claimed is:
 1. A lead assay comprising the steps of:(a)providing an aqueous solution suspected of containing lead; (b)isolating said lead from said solution in such a manner that said leadremains in solution: (c) introducing to said solution of step (b) anenhancing reagent that combines with said lead and prevents said leadfrom precipitating from said solution; followed by (d) introducing tosaid solution an enzyme the activity of which is inhibited in thepresence of lead and a substrate for said enzyme: and (e) measuring theamount of lead as a function of said activity of said enzyme.
 2. Thelead assay of claim 1 wherein the reagent which combines with lead instep (b) comprises a chelator having a lead equilibrium binding constantin the range of about 4 log K to aout 13 log K.
 3. The lead assay ofclaim 2 wherein the lead equilibrium binding constant is in the range ofabout 6 log K to about 9 log K.
 4. The lead assay of claim 2 wherein thechelator is selected from the group consisting of N-benzyliminodiaceticacid, ethylenebis(oxyethylenenitrilo) tetraacetic acid,ethylenediaminetetraacetic acid, L-histidine monohydrochloridemonohydrate, N-(2-hydroxyethyl)-iminodiacetic acid, iminodiacetic acid,DL-penicillamine, methyliminodiacetic acid, nitrilotriacetic acid,sodium citrate, and d-hydroxyquinoline-5-sulfonic acid hydrate.
 5. Alead assay comprising the steps of:(a) providing an aqueous solutionsuspected of containing lead, said solution having been separated fromcompounds that are affected by the presence of lead, said solutionfurther having been neutralized; (b) introducing into said solution alead chelator having a lead equilibrium binding constant in the range ofabout 4 log K to about 13 log K; followed by (c) introducing into saidsolution (i) an enzyme the activity of which is inhibited by lead and(ii) a substrate which reacts with the enzyme; (d) incubating thesolution of step (c): (e) stopping the incubation step after apredetermined interval; and (f) measuring the amount of lead as afunction of enzyme activity.
 6. The lead assay of claim 5 wherein theisolating step includes releasing lead from a sample of whole blood. 7.The lead assay of claim 5 wherein the method further includesneutralizing the sample solution before the enzyme incubating step. 8.The lead assay of claim 5 wherein the chelator has a lead equilibriumbinding constant in the range of about 6 log K to about 9 log K.
 9. Themethod of claim 5 wherein the chelator is selected from the groupconsisting of N-benzyliminodiacetic acid,ethylenebis(oxyethylenenitrilo) tetraacetic acid,ethylenediaminetetraacetic acid, L-histidine monohydrochloridemonohydrate, N-(2-hydroxyethyl)-iminodiacetic acid, iminodiacetic acid,DL-penicillamine, methyliminodiacetic acid, nitrilotriacetic acid,sodium citrate, and d-hydroxyquinoline-5-sulfonic acid hydrate.
 10. Themethod of claim 5 wherein the chelator is selected from the groupconsisting of 8-hydroxy-5-(2'-hydroxyphenylazo)quinoline,8-hydroxy-5-(phenylazo)quinoline, N-(2-carboxyphenyl)iminodiacetic acid,N-(acetonyl)iminodiacetic acid, N-(dithiocarboxy)aminoacetic acid,N,N-bis(2'-hydroxyethyl)glycine, and glycine.
 11. The method of claim 5wherein the chelator is selected from the group consisting ofdihydroxyphenyl acetic acid, N-(2'-carboxyethyl) iminodiacetic acid,dihydroxybenzoic acid, 3,4,dihydroxybenzene sulfonic acid, melonic acid,1-hydroxy-1-(3'-pyridyl) methane sulfonic acid, and4-aminopyridine-2,6-dicarboxylic acid.
 12. The lead assay of claim 5wherein the aqueous solution in step (b) is acidified and the leadchelator of step (c) is present in a neutralizing buffer such thatperforming step (c) results in bringing the acidified solution of step(b) to neutral pH.
 13. The lead assay of claim 5 wherein the enzyme isaminolevulinic acid dehydratase.
 14. The lead assay of claim 13 whereinthe enzyme is activated with a reducing agent selected from the groupconsisting of dithiothreitol, glutathione, mercaptoethanol and cysteine.15. The lead assay of claim 5 wherein the enzyme incubating stepincludes first incubating the sample solution in the presence ofaminolevulinic acid dehydratase and subsequently incubating the samplesolution in the presence of the substrate.
 16. The lead assay of claim15 wherein the substrate includes aminolevulinic acid.
 17. The leadassay of claim 5 wherein the stopping step includes adding a stopreagent.
 18. The lead assay of claim 17 wherein the stop reagentincludes HgCl₂.
 19. The lead assay of claim 5 wherein step (d) includesincubating the sample solution in the presence of a coloring reagent.20. The lead assay of claim 19 wherein the coloring reagent is selectedfrom the group consisting essentially of dimethylaminobenzaldehyde anddimethylaminocinnamaldehyde.
 21. An aqueous lead assay reagent solutionconsisting essentially of neutralizing buffer and a lead chelator havinga lead binding constant in the range of about 4 log K to about 13 log Kwherein the concentration of the chelator in the solution is in therange of 0.5 mM to 500 mM.
 22. The reagent solution of claim 21 whereinthe chelator has a lead equilibrium binding constant in the range ofabout 6 log K to about 9 log K.
 23. The reagent solution of claim 21wherein the chelator is selected from the group consisting ofN-benzyiminodiacetic acid, ethylenebis(oxyethylenenitrilo) tetraaceticacid, ethylenediaminetetraacetic acid, L-histidine monohydrochloridemonohydrate, N-(2-hydroxyethyl)-iminodiacetic acid, iminodiacetic acid,DL-penicillamine, methyliminodiacetic acid, nitrilotriacetic acid,sodium citrate, and d-hydroxyquinoline-5-sulfonic acid hydrate.
 24. Thereagent solution of claim 23 wherein the chelator is selected from thegroup consisting of 8-hydroxy-5-(2'-hydroxyphenylazo)quinoline,8-hydroxy-5-(phenylazo)quinoline, N-(2-carboxyphenyl)iminodiacetic acid,N-(acetonyl) iminodiacetic acid, N-(dithiocarboxy) aminoacetic acid,N,N-bis(2'-hydroxyethyl)glycine, and glycine.
 25. The reagent solutionof claim 21 wherein the chelator is selected from the group consistingof dihydroxyphenyl acetic acid, N-(2'-carboxyethyl) iminodiacetic acid,dihydroxybenzoic acid, 3,4,dihydroxybenzene sulfonic acid, melonic acid,1-hydroxy-1-(3'-pyridyl)methane sulfonic acid, and4-aminopyridine-2,6-dicarboxylic acid.
 26. A lead assay reagent kitcomprising:a container having a reagent solution consisting essentiallyof an aqueous neutralizing buffer and present therein a reagent which iscapable of forming a compound or complex with lead such that adding thesolution to an acidified aqueous sample containing lead will neutralizethe aqueous sample while preventing precipitation of lead therefrom; acontainer comprising an enzyme which is inhibited in the presence oflead; and a container comprising a substrate which reacts with saidenzyme.
 27. The lead assay reagent kit of claim 26 wherein the kitfurther includes:a container for a stop reagent for stopping thereaction between the substrate and the enzyme; a container for acoloring reagent for forming a chromophore upon reaction with a productof the substrate and enzyme reaction; and; a reducing reagent includedin either the substrate or enzyme containiner in an amount effective toincrease the activity of the enzyme reacting with the substrate.
 28. Thelead assay reagent kit of claim 27 wherein the reducing agent comprisesdithiothreitol, the enzyme comprises aminolevulinic acid dehydratase andthe substrate comprises aminolevulinic acid and the product of thesubstrate and enzyme comprises porphobilinogen.